Insulin is essential for proper metabolism in humans: in addition to its familiar role as the chief regulator of blood sugar levels in humans, it is essential for carbohydrate, lipid, and protein metabolism, as well. Pancreatic beta (β) cells of the islets of Langerhans, epithelial cells dispersed throughout the pancreas, secrete insulin. When β cells are destroyed or their function impaired, insulin production declines, and diabetes results.
Diabetes currently affects about 14 to 16 million individuals in the United States alone. Diabetes is a chronic condition characterized by an abnormally elevated plasma glucose level. The condition may result from an absolute deficiency of insulin due to the autoimmune destruction of insulin-secreting cells (i.e., Type I diabetes), or it may result from a relative deficiency of insulin due to either a secretory defect of production (i.e., by insulin-synthesizing cells) and/or by a resistance to the action of insulin (i.e., Type II diabetes).
For those patients that respond to insulin therapy, limitations remain in the methods of administering the hormone. For instance, insulin may be administered to a diabetic patient by way of an insulin pump. However, conventional insulin pumps deliver insulin to a patient at a set, constant rate (e.g., by pre-determined bolus size). Thus, the patient must constantly monitor his own blood glucose levels, by taking blood samples four or five times each day. Careful blood glucose monitoring is essential, since there is an ongoing risk of administering too much insulin, which may cause hypoglycemic shock. Hypoglycemic shock may cause a coma, and, not infrequently, may be fatal.
Drugs that promote insulin secretion or that lower glucose levels by other means are commonly prescribed to treat patients with type II diabetes. Sulfonylureas are the principal drugs prescribed to such patients. They stimulate insulin production by directly stimulating β cells; the effectiveness of such drugs therefore depends on the number of functioning β cells remaining in the pancreas. Repaglinide also stimulates insulin production by stimulating β cells, but differs structurally from the sulfonyluereas. Other drugs, such as metformin and troglitazone (known better by its brand name, REZULIN®), lower glucose levels by reducing glucose production in the liver and by promoting insulin sensitivity. Another drug, acarbose, inhibits digestive enzyme secretion and thereby delays digestion of carbohydrates (which when broken down in the body ultimately yield glucose). The efficacy of these drugs is tested first in vitro using existing cell lines that seek to model insulin-secreting β cells. None of these cell lines provides a satisfactory model, however, because they lose their responsiveness to glucose. As a result, in vitro studies of insulin-secreting drugs currently provides only limited information regarding their efficacy.
Understanding the function and development of insulin-secreting β cells is a critical step in developing better drugs to treat—and ultimately cure—diabetes. Pancreatic endocrine and exocrine cells (the cells that secrete insulin and other hormones) originate from a precursor epithelial cell during the development of the pancreas. G. Teitelman and J. K. Lee, “Cell lineage analysis of pancreatic islet cell development: glucagon and insulin cells arise from catecholarninergic precursor present in the pancreatic duct.” Dev. Biol. 121:454–466 (1987); R. L. Pictet et al., “An ultrastructual analysis of the developing embryonic pancreas.” Dev. Biol. 29:436–467 (1972) (the foregoing publications, and all other publications cited herein, are incorporated by reference in their entirety). Various differentiation factors are required to achieve the mature phenotype characteristic of islet β cells.
New β cells are formed from existing islets and from ductal epithelial cells. The latter source has greater intrinsic biological relevance. Indeed, the possibility of differentiating insulin-secreting cells from non-endocrine cells supports the hypothesis that the biological source (pancreatic ductal epithelium) for this compensatory mechanism may be present even in the setting of a generalized destruction of the entire population of islet β cells. This is strongly supported by recent studies demonstrating that primary cultures of epithelial ductal cells (from human and mouse pancreas) are susceptible to undergo differentiation into endocrine cells. V. K. Ramiya et al., “Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.” Nature Medicine, 6(3):278–82 (2000); S. Bonner-Weir et al., “In vitro cultivation of human islets from expanded ductal tissue. Proc. Nratl. Aca. Sci. USA, 14:7999–8004 (1997).
An incretin hormone, glucagon-like-peptide-1 (GLP-1), is believed to play a role in the development of the pancreas, though researchers have disagreed as to precisely what this role is. A decade ago, for example, U.S. Pat. No. 5,120,712, the entirety of which is incorporated by reference, stated that “The failure to identify any physiological role for GLP-1 caused some investigators to question whether GLP-1 was in fact a hormone and whether the relatedness between glucagon and GLP-1 might be artifactual.” Researchers have more recently learned that GLP-1 has a function in rats. Bonner-Weir et al., for example, demonstrated that an analog of the incretin hormone glucagon-like-peptide-1 (GLP-1), termed exendin-4, was able to increase islet mass in adult animals previously subjected to subtotal pancreatectomy. G. Xu et al., “Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats.” Diabetes 48:2270–2276 (1999). Similarly, one of the inventors has demonstrated that treating glucose-intolerant aging Wistar rats with GLP-1 restored normal glucose tolerance and induced islet cell proliferation. Y. Wang et al., “Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats.” J Clin Invest 99:2883–2889 (1997).
Islet duodenal homeobox-1 (“IDX-1,” also known variously as IPF-1/STF-1 and PDX-1) is a homeodomain protein and an insulin gene transcription factor expressed in the early pancreatic gland of the embryo. During pancreatic islet development, IDX-1 plays an important role in determining islet cell differentiation. It is the early IDX-1 gene expression during embryogenesis, coupled with the activation of other transcription factors (for example, NeuroDBeta 2, Pax 4, etc.), that determine the pancreatic endocrine hormone production. In adult (mature) animals, the expression of IDX-1 is repressed in the majority of pancreatic cells, with the exception of the β- and δ-cells (somatostatin-secreting cells) of the islets of Langerhans.
The mechanisms regulating proliferation and differentiation of the pancreatic hormone-producing cells and the chronology of these biological events are still largely undetermined. The sequence of events one of the inventors describes in U.S. patent application Ser. No. 09/920,868, filed Aug. 2, 2001, now U.S. Pat. No. 6,642,003, issued Nov. 4, 2003, suggests that the ability of regulating glucose uptake by the islet-specific glucose transporter GLUT2 is the first step necessary for the “sensitization” of the regulatory region(s) of the insulin gene to glucose. This would then promote the transcription of insulin mRNA. GLP-1-dependent activation of IDX-1 would further “commit” these cells toward a β cell-like pathway of differentiation by inducing the synthesis of glucokinase, the chief element of the “glucose-sensing machine” of the islets of Langerhans.
Researchers have learned much of the role of GLP-1 and IDX-1 in the rat and mouse, where knock-out mouse or other animal models were available to study the role of these hormones. Researchers know little of the role GLP-1 and IDX-1 in the development of human insulin-secreting cells, or of their interaction with other hormones present in the endocrine system. There is therefore an important need in the art for an analytical tool that permits researchers to elucidate the role of GLP-1 and IDX-1 in humans. A human model would be of immense importance in testing theories of endocrine development, in evaluating antidiabetic drugs, and developing new approaches to treat diabetes.
An important need exists in the art to implement the insulin-regulating abilities of GLP-1. Numerous disease conditions are related to the failed or deteriorated insulin regulation properties of particular cells in the body, such as diabetes; or the substantial lack of these cells in the body. Technology incorporating the insulin-regulating abilities of GLP-1 may obviate these disease conditions. A variety of potential insulin delivery applications may similarly be implemented in conjunction with this technology, such as insulin pumps which reside exterior to the body and implantable structures that release insulin internally in a glucose-dependent fashion.